CL-1724 Antibiotic compounds, their production and use

ABSTRACT

A strain of actinomycete, NRRL 15758, is capable of producing the CL-1724 complex of compounds under conditions of aerobic fermentation in the presence of assimilable sources of carbon and nitrogen. The compound CL-1724B-2 exhibiting antimicrobial and antitumor activity, is claimed together with pharmaceutical compositions and methods of treating microbial infections and tumors in mammals employing these pharmaceutical compositions.

BACKGROUND OF THE INVENTION

The present invention relates to pharmaceutical compounds, a purifiedstrain of microorganism capable of producing these compounds, and topharmaceutical compositions and methods of treatment. More particularly,the present invention relates to the compound CL-1724B-2 produced byfermentation, to a purified strain of actinomycete capable of producingthis compound, to pharmaceutical compositions containing the compound,and to a method of treating microbial infections employing thesecompositions.

BRIEF DESCRIPTION OF THE DRAWING

In the drawing:

FIG. 1 is the ultraviolet spectrum of CL-1724B-1 compound in methanol.

FIG. 2 is the ultraviolet spectrum of CL-1724B-2 compound in methanol.

FIG. 3 is the infrared spectrum of CL-1724B-1 compound in chloroform.

FIG. 4 is the infrared spectrum of CL-1724B-2 compound in chloroform.

FIG. 5 is the 300 MHz proton magnetic resonance spectrum of CL-1724B-2compound in deuterochloroform.

FIG. 6 is the 300 MHz proton magnetic resonance spectrum of CL-1724B-1compound in deuterochloroform.

SUMMARY AND DETAILED DESCRIPTION The Microorganism

In accordance with the present invention, the CL-1724 complex ofcompounds which possesses antimicrobial activity is produced bycultivating a purified strain of actinomycete, NRRL 15758, underartificial conditions until a sufficient quantity of the CL-1724 complexis formed, and subsequently isolating the individual components.

The actinomycete isolate suitable for the purposes of this invention wasfound in a soil sample collected in Florida, USA. The microorganism wasisolated from the soil sample by standard agar plating techniques usinga suitable agar medium containing salts such as potassium dihydrogenphosphate, potassium chloride, magnesium sulfate, ferrous sulfate, andcarbon sources such as glycerol and asparagine.

The soil sample was heated in a boiling water bath prior to being platedonto the agar medium and, once plated, was incubated at a favorabletemperature, particularly 33° C., to allow for the development of thesoil microorganisms.

The CL-1724 complex producing microorganism isolated from the soilsample by the procedure described above is an unidentified actinomycetewhich has been deposited with the Northern Regional Research Center ofthe United States Department of Agriculture, 1815 North UniversityStreet, Peoria, Ill. 61604, under the terms and conditions of theBudapest Treaty for the Deposit of Microorganisms where it has beengiven the designation NRRL 15758. This culture of microorganism is alsobeing maintained as a dormant culture in lyophile tubes, cryogenicvials, and in soil sample tubes in the Warner-Lambert/Parke-DavisCulture Collection, 2800 Plymouth Road, Ann Arbor, Mich. 48105, were itbears the designation WP-1234.

Actinomycete NRRL 15758 of the present invention has somecharacteristics in common with an actinomycete isolate namedActinomadura pulveraceus, ATCC 39100, described in European PatentApplication No. 0095154 published Nov. 30, 1983. It has been found,however, that the microorganism of the present invention, NRRL 15758,can be differentiated from isolate ATCC 39100 by several importantfeatures.

In Table 1 there is shown a comparison of the utilization of carbonsources by NRRL 15758 and ATCC 39100. Utilization of carbon sources bythe two microorganisms was determined by the methods detailed inPridham, T. G. and Gottlieb, D., J. Bacteriol., 56:107-114 (1965).

                  TABLE 1                                                         ______________________________________                                        Comparison of Carbon Source Utilization                                       of ATCC 39100 and NRRL 15758                                                               Utilization*                                                     Carbon Source  ATCC 39100 NRRL 15758                                          ______________________________________                                        Glucose        +          +                                                   Xylose         +          +                                                   Arabinose      -          +                                                   Rhamnose       +          +                                                   Fructose       -          -                                                   Galactose      ±       -                                                   Raffinose      -          -                                                   Mannitol       -          +                                                   Inositol       ±       -                                                   Salicin        -          -                                                   Sucrose        +          +                                                   ______________________________________                                         *+  = Utilization                                                             ± = Doubtful utilization                                                   - = No detectable utilization                                            

The microorganism of this invention, NRRL 15758, was able to utilizemannitol and arabinose, while ATCC 39100 was not. Additionally,differences between the two microorganisms in the utilization ofinositol and galactose were also noted.

As a result of these differences in the metabolic utilization of carbonby the two microorganisms, isolate NRRL 15758 is considered a newspecies of Actinomadura.

The Fermentative Production of the CL-1724 Complex of Compounds

The CL-1724 complex of compounds is produced during aerobic fermentationunder controlled conditions by isolate NRRL 15758. The fermentationmedium consists of sources of carbon, nitrogen, minerals, and growthfactors. Examples of suitable carbon sources are glycerol and varioussimple sugars such as glucose, mannose, fructose, xylose, ribose, andother carbohydrate-containing materials such as dextrin, starch,cornmeal, and whey. Normally, the quantity of carbon source materials inthe fermentation medium varies from about 0.1 to about 10 percent byweight.

Suitable nitrogen sources in the fermentation medium include organic,inorganic, and mixed inorganic-organic nitrogen-containing materials.Examples of such materials are cottonseed meal, soybean meal, corn germflour, corn steep liquor, distillers dry solubles, peanut meal,peptonized milk, and various ammonium salts.

The addition of minerals and growth factors is also helpful in theproduction of the CL-1724 complex. Examples of suitable minerals andgrowth factors include potassium chloride, sodium chloride, ferroussulfate, calcium carbonate, cobaltous chloride, zinc sulfate, andvarious yeast and milk products.

The preferred method for producing the CL-1724 complex of compounds isby the submerged culture fermentation method. According to this method,the fermentation medium is prepared by dissolving or suspending theingredients in water and subsequently sterilizing the resulting mediumby autoclaving or by steam heating. The sterilized medium is cooled to atemperature of between 16° C. and 45° C., inoculated with themicroorganism, and thereafter fermentation is carried out with aerationand agitation.

In the submerged culture method, fermentation is carried out inshake-flasks or in stationary tank fermentors. In shake-flasks, aerationis achieved by agitating the flasks to bring about intimate mixing ofthe inoculated medium with air. In stationary tank fermentors, agitationis provided by impellers which may take the form of disc turbines, vaneddiscs, or open turbine or marine propellers. Aeration is accomplished bysparging air or oxygen into the agitated mixture. Under theseconditions, adequate production of the CL-1724 complex is normallyachieved after a period of from about two to ten days.

In an alternative embodiment, the CL-1724 complex may also be producedby solid state fermentation of the microorganism.

The following illustrative examples of the fermentative production ofthe CL-1724 complex of compounds are provided to enable one skilled inthe art to carry out the present invention. These examples are not to beread as limiting the present invention as defined by the appendedclaims, but as merely illustrative thereof.

EXAMPLE 1 Isolation of Actinomycete NRRL 15758

The culture of actinomycete NRRL 15758 of the present invention,following its isolation from a CIM 41 agar plate, by the techniquedescribed above, was transferred to an agar slant employing CIM 23medium and incubated at 33° C. for seven to fourteen days.

                  TABLE 2                                                         ______________________________________                                         Formulation of CIM 41                                                        ______________________________________                                        K.sub.2 HPO.sub.4    1.0     g                                                MgSO.sub.4.7H.sub.2 O                                                                              0.5     g                                                KCl                  0.5     g                                                FeSO.sub.4.7H.sub.2 O                                                                              0.01    g                                                Glycerol             30.0    g                                                L-Asparagine         2.5     g                                                Agar                 15.0    g                                                Distilled Water      1000.0  ml                                               ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                         Formulation of CIM 23 Medium                                                 ______________________________________                                        Amidex corn starch    10.0    g                                               N-Z amine, Type A     2.0     g                                               Beef Extract (Difco)  1.0     g                                               Yeast Extract (Difco) 1.0     g                                               CoCl.sub.2.6H.sub.2 O 0.02    mg                                              Agar                  20.0    g                                               Distilled water       1000.0  ml                                              ______________________________________                                    

EXAMPLE 2 Shake-Flask Fermentation

A portion of the microbial growth from the agar slant in Example 1 wasused to inoculate an 18-mm×150-mm seed tube containing 5 ml of SD-05seed medium. The inoculated seed was shaken at 33° C. for three days.

                  TABLE 4                                                         ______________________________________                                         Formulation of SD-05 Seed Medium                                             ______________________________________                                        Yeast extract (Amberex 1003, Amber Labs)                                                                5.0     g                                           Glucose monohydrate (Cerelose, Corn Products)                                                           1.0     g                                           Dextrin (Amidex B411, American Maize)                                                                   24.0    g                                           Casein digest (N-Z-case, Sheffield)                                                                     5.0     g                                           Spray dried meat solubles (Jen-Kim)                                                                     3.0     g                                           Calcium carbonate         2.0     g                                           Deionized water           1000.0  ml                                          ______________________________________                                    

A 1-ml portion of the microbial growth from the seed tube wastransferred to a 300-ml baffled shake-flask containing 50 ml of SM-41production medium.

                  TABLE 5                                                         ______________________________________                                         Formulation of SM-41 Medium                                                  ______________________________________                                        Sucrose               15.0    g                                               Dextrin               10.0    g                                               Cotton seed meal      6.5     g                                               Peptonized milk       3.5     g                                               Torula yeast          2.5     g                                               Deionized water       1000.0  ml                                              ______________________________________                                    

The inoculated flask was incubated at 33° C. for four days with shaking(180 rpm gyratory shaker, 5-cm throw). Production of the CL-1724 complexwas observed for the first time in this broth.

To confirm the microbial production of the CL-1724 complex, a second50-ml batch of SM-41 production medium, contained in a 300-ml baffledshake-flask, was inoculated with 2 ml of microbial growth from the seedtube. This mixture was incubated at 33° C. for four days with shaking(180 rpm gyratory shakes, 5-cm throw).

The antitumor activity of both fermentation broths was assayed usingL1210 mouse leukemia cells grown in tissue culture. The assay techniqueis fully described in Cancer Chemotherapy Reports, Part 3, Vol. 3, No. 2(1972), Deran, Greenberg, MacDonald, Schumacher and Abbott. A resultingL1210 leukemia cell growth of 0 to 35% in the presence of the CL-1724complex was considered active with 0% as most active. The observedactivity of the fermentation broths of Example 2, at a dilution of1:100, is given in Table 6.

                  TABLE 6                                                         ______________________________________                                        Antitumor Activity of Fermentation Broths                                     from Example 2 (As Measured Against                                           L1210 Mouse Leukemia Cells)                                                                % L1210 Cell Growth                                                                       Freeze-Dried                                         Fermentation Stage                                                                           Supernate Ethanol Extract                                      ______________________________________                                        a. Shake-flask 1                                                                             18        26                                                   b. Shake-flask 2                                                                             12        --                                                   ______________________________________                                    

The crude fermentation broth of shake-flask 2 was also tested forantibacterial activity against various organisms using the agar-discmethod. The crude broth was found to be active against Alcaligenesviscolactis, Bacillus subtilis, Micrococcus luteus, M. lysodeikticus,Branhamella catarrhalis, and Staphylococcus aureus. The antimicrobialactivity of the shake-flask 2 fermentation broth as measured by theagar-disc diffusion assay is shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        Antimicrobial Activity of the                                                 Shake-Flask 2 Fermentation Broth                                                                           Zone Diameter                                    Microorganism  Culture Number                                                                              (mm)                                             ______________________________________                                        Aerobacter aerogenes                                                                         0126          0                                                Alcaligenes faecalis                                                                         ATCC 8750     0                                                A. viscolactis ATCC 21698     18.5                                            Escherichia coli                                                                             ATCC 10536    0                                                Bacillus subtilis                                                                            04555          16.5                                            B. subtilis    04969          18.0                                            B. subtilis    04969          18.5                                            Micrococcus luteus                                                                           05064          20.0                                            M. lysodeikticus                                                                             04783          21.0                                            Branhamella catarrhalis                                                                      03596          28.0                                            Staphylococcus aureus                                                                        02482          17.0                                            Pseudomonas aeruginosa                                                                       NCTC 10490    0                                                Klebsiella pneumoniae                                                                        05037         0                                                Xanthomonas phaseoli                                                                         06002         0                                                Proteus vulgaris                                                                             05062         0                                                Kloeckera africana                                                                           1570          0                                                K. brevis      1378          0                                                Rhodotorula glutinis                                                                         M1384         0                                                Saccharomyces cerevisiae                                                                     01525         0                                                Torulopsis albida                                                                            M1390         0                                                Penicillium avellaneum                                                                       M2988         0                                                ______________________________________                                    

EXAMPLE 3 30-Liter Stirred-Jar Fermentation

A cryogenic vial containing approximately 1 ml of a suspension of theculture was used to inoculate 600 ml of SD-05 seed medium contained in a2-liter baffled shake-flask. The inoculated flask was incubated forabout 76 hours at 33° C. on a gyratory shaker at 130 rpm.

After about 76 hours, the contents of the seed flask were transferredaseptically to a 30-liter stainless steel fermentor containing 16 litersof PM-11 production medium. The inoculated medium was incubated at 33°C. for six days while being stirred at 300 rpm and sparged with air at arate of 1 volume air/volume medium/minute.

The production of the CL-1724 complex was monitored throughout thefermentation cycle by in vitro assay against L1210 mouse leukemia cellsand by antimicrobial activity against M. luteus. In addition, suchfermentation parameters as pH and percent sedimentation were recordedthroughout the fermentation cycle. The data are presented in Table 8.

                  TABLE 8                                                         ______________________________________                                        Fermentation Profile and Bioactivity                                          of a 30-Liter Fermentation (Example 3)                                                    Bioactivity                                                       Fermen-               Zone                                                    tation       Percent  (mm)                                                    Time         Sediment vs. M.                                                                              L1210, % Growth                                   (Hours)                                                                              pH    (Growth) luteus                                                                              1:500                                                                              1:2500                                                                              1:5000                                                                              1:10000                          ______________________________________                                         0     6.8   --       --    --   --    --    --                               24     7.3   6.0       0    NA*  NA    NA    NA                               50     7.7   7.4      18     7.5 20    NA    NA                               73     7.9   7.4      22    0    0     17.8  NA                               96     8.0   6.7      20    0    0     0     27.2                             121    8.1   9.3      20    0    0     0     21.3                             144    8.1   10.7     20    0    0     16.8  27.6                             ______________________________________                                         *NA = not active                                                         

The harvested beer was also tested for antitumor activity against P388murine lymphocytic leukemia in vivo by the method detailed in thereference cited above in Example 2. The results of these tests arepresented in Table 9.

                  TABLE 9                                                         ______________________________________                                        Antitumor Activity of Fermentation                                            Broth from Example 3 Against P388                                             Murine Lymphocytic Leukemia In Vivo                                           Dilution of        % T/C*                                                     Fermentation Beer  Test 1  Test 2                                             ______________________________________                                        undiluted          123     164                                                1:2                132     142                                                1:4                132     136                                                1:8                126     133                                                ______________________________________                                         ##STR1##                                                                 

                  TABLE 10                                                        ______________________________________                                         Formulation of PM-11                                                         ______________________________________                                        Lactose                10.0    g                                              Sucrose                15.0    g                                              MgSO.sub.4.7H.sub.2 O  2.5     g                                              CaCO.sub.3             1.5     g                                              NaCl                   1.0     g                                              Spray dried whey (Krafen)                                                                            5.0     g                                              Defatted corn germ flour                                                                             7.5     g                                              Deionized water        1000.0  ml                                             ______________________________________                                    

EXAMPLE 4 200-Gallon Fermentation

Two cryogenic vials, each containing about 1 ml of a suspension of theNRRL 15758 culture, were used to inoculate two 2-liter seed flasks. Each2-L flask contained 600 ml of SD-05 medium. The inoculated flasks wereincubated at 33° C. with shaking on a gyratory shaker, 130 rpm. After 75hours of incubation, the contents of each seed flasks were used toinoculate two 30-liter stainless steel seed jars. Each seed jarcontained 16 liters of SD-05 medium. After inoculation, fermentation wascarried out at 33° C. for 24 hours, stirred at 300 rpm, and sparged withair at a rate of 1 volume air/volume medium/minute.

The growing microorganisms from the two seed fermentors were used toinoculate a 200-gallon (757-liter) production fermentor. The productionfermentor contained 155 gallons (587 liters) of PM-11 production mediumwhich was steam sterilized for 40 minutes at 121° C. The medium wascooled to 33° C. and then inoculated with about 30 liters of the growingmicroorganisms from the two seed jars. Fermentation was carried out forfour days at 33° C., stirred at 155 rpm, and sparged with air at 0.75volume air/volume medium/minute. A silicone-based antifoam was used tocontrol foaming as needed.

The bioactivity of the fermentation broth in the 200-gallon fermentorwas monitored throughout the fermentation cycle by in vitro assayagainst L1210 mouse leukemia cells and by antimicrobial activity againstM. luteus. The data are presented in Table 11.

                                      TABLE 11                                    __________________________________________________________________________    Fermentation Profile and Bioactivity of a                                     200-Gallon Fermentation (Example 4)                                           Fermentation                                                                           Percent                                                                            Bioactivity                                                     Time     Sediment                                                                           Zone (mm) vs.                                                                         L1210, % Growth                                         (Hours)                                                                              pH                                                                              (Growth)                                                                           M. luteus                                                                             1:100                                                                            1:500                                                                            1:2500                                                                            1:5000                                                                            1:10000                                   __________________________________________________________________________     0     7.0                                                                             --   --      -- -- --  --  --                                        12     6.8                                                                             4.0  0       -- -- --  --  --                                        24     7.1                                                                             6.7  0       NA*                                                                              NA NA  --  --                                        36     7.5                                                                             6.0  0       NA NA NA  --  --                                        48     7.8                                                                             7.4  0       13.3                                                                             NA NA  --  --                                        60     7.7                                                                             8.0  16.0    11.5                                                                             NA NA  --  --                                        72     7.8                                                                             7.4  19.0    0  0  11.5                                                                              NA  --                                        84     7.7                                                                             8.0  19.0    -- 0  0   17.0                                                                              NA                                        96     7.8                                                                             8.0  20.0    -- 0  0   11.9                                                                              NA                                        __________________________________________________________________________     *NA = not active                                                         

EXAMPLE 5 2000-Gallon Fermentation

A cryogenically preserved sample of isolate NRRL 15758 was thawed and a1-ml sample was used to inoculate 600 ml of SD-0.5 medium contained in aa 2-liter Erlenmeyer seed flask. The inoculated flask was incubated for72 hours at 33° C. while being shaken on a gyratory shaker, 130 rpm(5-cm throw).

The resulting microbial growth was used to inoculate 16 liters of SD-05seed medium in a 30-liter stirred-jar fermentor. THe inoculated jar wasincubated for 24 hours while being stirred at 300 rpm and sparged withair at a rate of 1 volume air/volume medium/minute.

The microbial growth from the stirred-jar fermentor was used toinoculate 75 gallons (284 liters) of SD-05 seed medium contained in a200-gallon (757-liter) fermentor. The medium was sterilized by steamheating at 121° C. for 40 minutes, cooled to 33° C., and then inoculatedwith about 16 liters of the microbial growth from the stirred-jar. Theinoculated 200-gallon fermentor was incubated for 40 hours at 33° C.while being stirred at 155 rpm and sparged with air at a rate of 0.75volume air/volume medium/minute.

The microbial growth from the 200-gallon seed fermentor was used toinoculate a 2000-gallon (7570-liter) production fermentor. Theproduction fermentor contained about 1300 gallons (4920 liters) of PM-11production medium which was sterilized by steam heating for 40 minutesat 121° C. After sterilization the fermentor was cooled to 33° C.,inoculated with about 75 gallons of microbial growth from the 200-gallonseed fermentor, incubated for 96 hours with stirring at 125 rpm, andsparged with air at a rate of 0.75 volume air/volume medium/minute.

The production of the CL-1724 complex was monitored throughout thefermentation cycle by in vitro assay against L1210 mouse leukemia cellsand M. luteus. In addition, fermentation parameters such as pH andpercent sedimentation were recorded. The data are presented in Table 12.

                                      TABLE 12                                    __________________________________________________________________________    Fermentation Profile and Bioactivity of a                                     2000-Gallon Fermentor (Example 5)                                             Fermentation                                                                           Percent                                                                            Bioactivity                                                     Time     Sediment                                                                           Zone (mm) vs.                                                                         L1210, % Growth                                         (Hours)                                                                              pH                                                                              (Growth)                                                                           M. luteus                                                                             1:100                                                                            1:500                                                                            1:2500                                                                            1:5000                                                                            1:10000                                   __________________________________________________________________________     0     6.9                                                                             --   --      -- -- --  --  --                                        12     6.9                                                                             6.0  --      -- -- --  --  --                                        24     7.2                                                                             6.7   0      NA*                                                                              NA NA  --  --                                        36     7.4                                                                             6.7   0      12.1                                                                             NA NA  --  --                                        48     7.7                                                                             7.4  19      0  0  2.5 --  --                                        60     7.8                                                                             9.3  21      0  0  2.2 --  --                                        72     7.7                                                                             10.0 21      0  0  0   0   8.4                                       78     7.8                                                                             9.3  21      0  0  0   0   11.9                                      84     7.8                                                                             10.0 21      -- 0  0   0   7.2                                       96     7.9                                                                             9.3  20      -- 0  0   0   10.6                                      __________________________________________________________________________     *NA = not active                                                         

Chemical Isolation of the CL-1724 Complex

Fermentation beer (625 liters) prepared as described above in Example 4was adjusted to pH 6.2 with sulfuric acid and mixed for one hour with568 liters of ethyl acetate. Celite 545 (22.7 kg) was added and themixture was filtered through a 46-cm plate-and-frame filter press. Thefiltrate was allowed to stand and the lower aqueous layer whichseparated was removed. The filter cake was washed with 284 liters offresh ethyl acetate and the wash was used to extract the separatedaqueous layer described above.

After allowing the mixture to separate, the lower aqueous layer wasremoved. The two ethyl acetate extracts were combined and concentratedto 28.5 liters. After removal of a small amount of inactive precipitatedsolids, the ethyl acetate extract was further concentrated to 1.6liters.

The ethyl acetate concentrate was diluted to 16 liters with heptane, andthe mixture was extracted twice with 16 liters of with methanol-water(90:10). The lower aqueous methanol layers were combined, washed with 16liters of heptane, and then concentrated to 8 liters. After furtherextraction with 8 liters of hexane-chloroform (2:1), the aqueousmethanol concentrate (which contained the majority of the CL-1724complex) was separated and concentrated to 2 liters. Furtherconcentration was effected by adding absolute ethyl alcohol in portionsand evaporating to dryness to yield 13.1 g of crude CL-1724 complex.

The crude complex isolated by the methods detailed above was dissolvedin methanol and the resulting solution diluted with ethyl acetate.Concentration with addition of ethyl acetate allowed the removal of themethanol to yield two liters of an ethyl acetate soluble fraction andabout 1 g of insoluble material which was removed by filtration.

The ethyl acetate-soluble fraction was divided into three portions andeach portion was chromatographed separately over silica gel(Prep-PAK-500 (TM), Waters Associates, Milford, Mass., USA). Stepgradient elution was effected by employing first, chloroform (2 liters),then chloroform-methanol (96:4, 4 liters), next chloroform-methanol(9:1, 2 liters), and finally chloroform-methanol (8:2, 2 liters). TheCL-1724 complex was eluted in the last 3.5 liter portion of the 96:4chloroform-methanol fractions. Each fraction was analyzed by thin-layerchromatographic assay on silica gel (Baker-flex silica gel, 1B2-Fflexible sheets, J. T. Baker, Co., Phillipsburg, N.J., USA) employing1-butanol-ethyl acetate-n-heptane-ethanol-water (2:8:4:3:1) as a mobilephase. Detection was by bioautography versus Micrococcus luteus. Basedupon this analysis, fractions were combined and concentrated to drynessto yield residues weighing 1.4 g, 0.6 g, and 0.35 g for fractions A, B,and C, respectively.

Purification of the CL-1724 complex was achieved by furtherchromatography on silica gel. Thus, a 1.02 g portion of fraction A fromabove was dissolved in dichloromethane-methanol (99:1) andchromatographed over 75 g of silica gel (Waters Associates, Milford,Mass., USA) contained in a 26 mm×380 mm glass column. Elution waseffected with dichloromethane containing increasing amounts of methanolvia a step gradient. Each fraction was analyzed by high pressure liquidchromatographic assay and using system b described in Table 13.Fractions richest in the desired materials were combined andconcentrated. Elution with dichloromethane-methanol (97.5:2.5) afforded23.5 mg of semipurified CL-1724B-1, while elution withdichloromethane-methanol (97:3) yielded 25.9 mg of semipurifiedCL-1724B-2.

Purification and Properties of CL-1724B-1

An 18 mg portion of semipurified CL-1724B-1 from above was dissolved in1 ml of methanol and chromatographed over 125 g of C-18 silica gel (40μm particle size, Whatman Chemical Separation, Inc., Clifton, N.J., USA)packed in a 26 mm×460 mm glass column. The silica gel was prepared forchromatography by washing with methanol and then equilibrating with amobile phase consisting of 0.1M sodium acetate buffer (pH4.0)-methanol-acetonitrile (40:30:30). After application of the charge,the column was eluted with the same buffer-methanol-acetonitrile solventsystem. Several fractions were collected and each was assayed by highpressure liquid chromatography using system a described in Table 13.Fractions containing CL-1724B-1 as the only UV-absorbing peak werecombined. The combined eluates (370 ml) were extracted once with 1500 mlof dichloromethane, and then again with 1000 ml of dichloromethane. Thecombined extracts were washed with 300 ml of water and then concentratedto dryness to yield 5.0 mg of chromatographically pure CL-1724B-1. Theproperties of CL-1724B-1 are listed in Table 13.

                  TABLE 13                                                        ______________________________________                                         Properties of CL-1724B-1                                                     ______________________________________                                        Ultraviolet absorption                                                        in methanol      max          a                                                                318 nm        8.60                                                            280 nm       11.94                                                            252 nm       19.18                                                            204 nm       23.58                                           Infrared absorption                                                                            Principal absorption peaks at                                spectrum in chloroform                                                                         2940, 1735, 1685, 1615, 1600,                                                 1530, 1525, 1465, 1455, 1375,                                                 1315, 1255, 1160, 1080, 1025,                                                 990, 910, 880, and 855                                                        reciprocal centimeters.                                      300 MHz proton magnetic                                                                        Principal signals at 0.90                                    resonance spectrum in                                                                          (multiplet), 1.25 (singlet),                                 deuterochloroform                                                                              1.35 (multiplet), 1.50-2.25                                                   (multiplet), 2.35 (multi-                                                     plet), 2.45-2.90 (multiplet),                                                 3.35-4.20 (multiplet), 4.25                                                   (multiplet), 4.60 (doublet),                                                  4.65 (multiplet), 5.00                                                        (doublet), 5.40 (singlet),                                                    5.50 (multiplet), 5.70                                                        (multiplet), 5.80 (doublet),                                                  5.95 (doublet), 6.25                                                          (singlet), 6.60 (multiplet),                                                  7.50 (singlet), 8.60                                                          (singlet), 12.75 (singlet),                                                   all signals measured in parts                                                 per million downfield from                                                    tetramethylsilane.                                           High pressure liquid                                                          chromatography                                                                System a:        Column: Partisil 10 ODS-3                                                     C-18 silica gel (Whatman                                                      Chemical Separation, Inc.,                                                    Clifton, New Jersey, USA),                                                    4.6 mm I.D. × 25 cm.                                                    Solvent: 0.05 M pH 7.1                                                        ammonium phosphate buffer-                                                    acetonitrile (45:55).                                                         Flow rate: 2.0 ml/min.                                                        Detection: Ultraviolet                                                        absorption at 254 nm.                                                         Retention time: 8.8 min.                                     System b:        Column: μ Porasil silica gel                                               (Waters Associates, Inc.,                                                     Milford, Massachusetts, USA),                                                 3.9 mm I.D. × 30 cm.                                                    Solvent: Dichloromethane-                                                     methanol (96:4).                                                              Flow rate: 1.5 ml/min.                                                        Detection: Ultraviolet                                                        absorption at 254 nm.                                                         Retention time: 4.6 min.                                     Thin-layer chroma-                                                            tography                                                                      System a:        Solid phase: Bakerflex IB2-F                                                  silica gel (J. T. Baker Co.,                                                  Phillipsburg, New Jersey,                                                     USA).                                                                         Mobile phase: 1-Butanol-                                                      ethyl acetate-n-heptane-                                                      ethanol-water (2:8:4:3:1).                                                    Detection: Bioautography                                                      vs. Micrococcus luteus.                                                       Rf: 0.45                                                     System b:        Solid phase: Silica gel 60                                                    F-254 (Merck & Co., Rahway,                                                   New Jersey, USA).                                                             Mobile phase: Chloroform-                                                     methanol (92:8).                                                              Detection: p-Anisaldehyde                                                     spray reagent.                                                                Rf: 0.40                                                     ______________________________________                                    

Purification and Properties of CL-1724B-2

Semipurified CL-1724B-2 was purified by chromatography over C-18 silicagel (40 μm particle size, Whatman Chemical Separation, Inc., Clifton,N.J., USA) in a manner similar to that described above for CL-1724B-1.Thus, application of a 1 ml solution of semipurified CL-1724B-2 (23 mg)in methanol to the above-described column (previously washed withmethanol and equilibrated with the buffered mobile phase), followed byelution with 0.1M sodium acetate buffer (pH 4.0)-methanol-acetonitrile(40:30:30), afforded fractions containing CL-1724B-2 as the onlyUV-absorbing component as determined by high pressure liquidchromatography assay. These fractions, totaling 630 ml in volume, werecombined and extracted once with 2500 ml of dichloromethane and againwith 1200 ml of dichloromethane. The combined extracts were washed with650 ml of water and concentrated to dryness to afford 8.6 mg ofchromatographically pure CL-1724B-2. The properties of CL-2724B-2 arelisted in Table 14.

                  TABLE 14                                                        ______________________________________                                         Properties of CL-1724B-2                                                     ______________________________________                                        Molecular weight 1374 Atomic mass units                                       Elemental analysis* 52.40% C, 6.26% H, 4.08% N, 9.32% S                       Ultraviolet absorption                                                        in methanol      max          a                                                                318 nm        8.12                                                            280 nm       11.16                                                            251 nm       18.64                                                            204 nm       29.84                                           Infrared absorption                                                                            Principal absorption peaks at                                spectrum in chloroform                                                                         3520, 2940, 1760, 1720, 1685,                                                 1600, 1580, 1520, 1450, 1385,                                                 1375, 1310, 1250, 1155, 1075,                                                 1015, 1000, 955, 900, 875,                                                    and 850 reciprocal centimeters.                              300 MHz proton magnetic                                                                        Principal signals at 0.90                                    resonance spectrum in                                                                          (multiplet), 1.25 (singlet),                                 deuterochloroform                                                                              1.40 (multiplet), 1.45-2.20                                                   (multiplet), 2.30 (multi-                                                     plet), 2.40-2.90 (multiplet),                                                 3.30-4.30 (multiplet), 4.50                                                   (multiplet), 4.70 (doublet of                                                 doublets), 4.95 (doublet),                                                    5.35 (singlet), 5.45                                                          (doublet), 5.70 (multiplet),                                                  5.80 (doublet), 5.90                                                          (doublet), 6.25 (singlet),                                                    6.55 (multiplet), 7.75                                                        (singlet), 8.65 (singlet),                                                    12.95 (singlet), all signals                                                  measured in parts per million                                                 downfield from tetramethyl-                                                   silane.                                                      High pressure liquid                                                          chromatogrpahy                                                                System a:        Column: Partisil 10 ODS-3                                                     C-18 silica gel (Whatman                                                      Chemical Separation, Inc.,                                                    Clifton, New Jersey, USA),                                                    4.6 mm I.D. × 25 cm.                                                    Solvent: 0.05 M pH 7.1                                                        ammonium phosphate buffer-                                                    acetonitrile (45:55).                                                         Flow rate: 2.0 ml/min.                                                        Detection: Ultraviolet                                                        absorption at 254 nm.                                                         Retention time: 10.0 min.                                    System b:        Column: μ Porasil silica gel                                               (Waters Associates, Inc.,                                                     Milford, Massachusetts, USA),                                                 3.9 mm I.D. × 30 cm.                                                    Solvent: Dichloromethane-                                                     methanol (96:4).                                                              Flow rate: 1.5 ml/min.                                                        Detection: Ultraviolet                                                        absorption at 254 nm.                                                         Retention time: 5.2 min.                                     Thin-layer chroma-                                                            tography                                                                      System a:        Solid phase: Bakerflex IB2-F                                                  silica gel (J. T. Baker Co.,                                                  Phillipsburg, New Jersey,                                                     USA).                                                                         Mobile phase: l-Butanol-                                                      ethyl acetate-n-heptane-                                                      ethanol-water (2:8:4:3:1).                                                    Detection: Bioautoography                                                     vs. Micrococcus luteus.                                                       Rf: 0.45                                                     System b:        Solid phase: Silica gel 60                                                    F-254 (Merck & Co., Rahway,                                                   New Jersey, USA).                                                             Mobile phase: Chloroform-                                                     methanol (92:8).                                                              Detection: p-Anisaldehyde                                                     spray reagent.                                                                Rf: 0.33                                                     ______________________________________                                         *By highresolution mass spectrometry.                                    

Biological Activity of CL-1724B-1 and CL-1724B-2

The biological activity of the compound of this invention against fivespecies of gram-negative bacteria, seven species of gram-positivebacteria, four species of yeast, and two species of fungi was determinedusing the microtiter dilution technique. This method is described by T.B. Conrath, "Handbook of Microtiter Procedures," Dynatech Corp.,Cambridge, Mass., USA (1972); and T. L. Gavan and A. L. Barry,"Microdilution Test Procedures" in Manual of Clinical Microbiology, E.H. Lennett ed., American Soc. for Microbiol., Washington, D.C., USA(1980).

Each agent is suspended in a nonaqueous solvent for several minutes tosterilize the compound or, if the compound is completely soluble inwater, the aqueous solution is sterilized by passage through a 0.2 to0.45 micron membrane filter.

Each well of a sterile 96-well microdilution tray is filled underaseptic conditions with 0.1 ml of Mueller-Hinton broth for antibacterialtests, and yeast extract-peptone-dextrose or buffered, supplementedyeast nitrogen base for tests using yeasts or fungi.

A 0.5 ml sample of the test compound solutions is added to each of theeight wells in the first row. A microdilutor apparatus is used tosimultaneously mix the contents of these wells and to transfer aliquotsto each succeeding row of cells to obtain a range of serially dilutedsolutions, e.g., concentrations of 1000, 333, 111, 37, 12.3, 4.1, 1.37,and 0.46 micrograms/ml. The last row of wells is untreated and serves asa control.

Each well containing broth and test compound is inoculated with aboutten microliters of inoculum of the test microorganism. One well in thelast row of wells (which are free of test compound) is not inoculated toprovide a sterility control. The trays are sealed and incubated. Mediainoculated with bacteria are incubated at 37° C. for 16-24 hours, whilethose containing yeasts or fungi are incubated at 28° C. for 36-48hours. During incubation, the inoculated medium is shaken at 100-140 rpmto increase contact between the cells and test compounds.

After the incubation period, each plate is placed on a test readingmirror and the inhibition end points are observed and recorded. Thelowest concentration of test compound producing inhibition of the growthof the microorganism (MIC value) is used as the measure of the activityof the test compound. MIC values of <0.5 μg/ml to 333 μg/ml areconsidered indicative of activity; MIC values of 333 μg/ml to 1000 μg/mlare considered indicative of marginal activity; and MIC values of >1000μg/ml are considered indicative of inactivity of the test compoundagainst the given microorganism.

The antimicrobial activities of CL-1724B-1 and CL-1724B-2 appear inTables 15 and 16, respectively.

                  TABLE 15                                                        ______________________________________                                        Antimicrobial Activity of CL-1724B-1                                                           Minimal Inhibitory                                                            Concentration                                                Microorganism    (μg/ml)    Rating                                         ______________________________________                                        Escherichia coli  0.06         Active                                         Salmonella typhimurium                                                                         <0.02         Active                                         Alcaligenes viscolactis                                                                        <0.02         Active                                         Branhamella catarrhalis                                                                        <0.02         Active                                         Pseudomonas aeruginosa                                                                          0.5          Active                                         Micrococcus luteus                                                                             <0.02         Active                                         Staphylococcus aureus                                                                          <0.02         Active                                         Streptococcus pyogenes                                                                         <0.02         Active                                         Streptococcus pneumoniae                                                                       <0.02         Active                                         Streptococcus faecalis                                                                         <0.02         Active                                         Bacillus cereus  <0.02         Active                                         Bacillus megaterium                                                                            <0.02         Active                                         Saccharomyces cerevisiae                                                                       <0.02         Active                                         Torulopsis albida                                                                              <0.02         Active                                         Mucor paraciticus                                                                               0.5          Active                                         Rhizopus japonicus                                                                              0.5          Active                                         ______________________________________                                    

                  TABLE 16                                                        ______________________________________                                        Antimicrobial Activity of CL-1724B-2                                                           Minimal Inhibitory                                                            Concentration                                                Microorganism    (μg/ml)    Rating                                         ______________________________________                                        Escherichia coli  0.05         Active                                         Salmonella typhimurium                                                                         <0.02         Active                                         Alcaligenes viscolactis                                                                        <0.02         Active                                         Branhamella catarrhalis                                                                        <0.02         Active                                         Pseudomonas aeruginosa                                                                          1.5          Active                                         Micrococcus luteus                                                                             <0.02         Active                                         Staphylococcus aureus                                                                          <0.02         Active                                         Streptococcus pyogenes                                                                         <0.02         Active                                         Streptococcus pneumoniae                                                                       <0.02         Active                                         Streptococcus faecalis                                                                         <0.02         Active                                         Bacillus cereus  <0.02         Active                                         Bacillus megaterium                                                                            <0.02         Active                                         Saccharomyces cerevisiae                                                                        0.17         Active                                         Torulopsis albida                                                                               0.17         Active                                         Mucor paraciticus                                                                               4.6          Active                                         Rhizopus japonicus                                                                             14            Active                                         ______________________________________                                    

The data appearing in Tables 15 and 16 indicate that CL-1724B-1 andCL-1724B-2 possess considerable activity as agents against a wide rangeof gram-negative and gram-positive bacteria, and fungi.

Cytotoxic Activity of CL-1724B-1 and CL-1724B-2 Against L1210 MurineLeukemia Cells in Vitro

The cytotoxic antitumor activity of CL-1724B-1 and CL-1724B-2 againstL1210 murine leukemia cells in vitro was determined using the methoddetailed in R. I. Geran, et al., "Protocols for Screening ChemicalAgents and Natural Products Against Animal Tumors and Other BiologicalSystem," 3rd Edition, Cancer Chemotherapy Reports, Part 3, Vol. 3, 1-87(1972) which is incorporated herein by reference. The data from thesetests appear in Table 17.

                  TABLE 17                                                        ______________________________________                                        Cytotoxic Activity of CL-1724B-1 and CL-1724B-2                               Against L1210 Murine Leukemia Cells in Vitro                                                ID.sub.50                                                       Compound      (μg/ml)                                                      ______________________________________                                        CL-1724B-1    1.18 × 10.sup.-4                                          CL-1724B-2     2.1 × 10.sup.-4                                          ______________________________________                                    

The in vivo antitumor activities of CL-1724B-1 and CL-1724B-2 againstP388 murine leukemia were tested in accordance with the protocol inGeran, et al. cited above. The mice were infected intraperitoneally onDay 0 and then administered appropriate doses of CL-1724B-1 orCL-1724B-2 on Days 1-9. The percent median survial times of treated micecompared to untreated mice (%T/C values) appear in Table 18.

                  TABLE 18                                                        ______________________________________                                        In Vivo Activity of CL-1724B-1 and CL-1724B-2                                 Against P388 Leukemia in Mice                                                                Dosage                                                         Compound       (mg/kg/inj)                                                                              % T/C*                                              ______________________________________                                        CL-1724B-1     0.020      toxic                                                              0.010      toxic                                                              0.005       93                                                                 0.0025    143                                                                 0.00125   127                                                 CL-1724B-2     0.020      140                                                                0.010      134                                                                0.005      125                                                                 0.0025    107                                                 ______________________________________                                         ##STR2##                                                                 

The compounds of the present invention are useful for theirantimicrobial activity as pharmaceutical compositions in combinationwith a compatible pharmaceutically acceptable carrier. Thesecompositions may also contain other antimicrobial agents. Thecompositions may be made up in any pharmaceutically appropriate form forthe desired route of administration. Examples of such forms includesolid forms for oral administration as tablets, capsules, pills, powdersand granules, liquid forms for topical or oral administration assolutions, suspensions, syrups, and elixirs, and forms suitable forparenteral administration such as sterile solutions, suspensions, oremulsions.

For preparing pharmaceutical compositions from the compounds describedby this invention, inert, pharmaceutically acceptable carriers can beeither solid or liquid. Solid form preparations include powders,tablets, dispersible granules, capsules, cachets, and suppositories. Asolid carrier can be one or more substances which may also act asdiluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, or tablet disintegrating agents; it can also be encapsulatingmaterial. In powders, the carrier is a finely divided solid which is inadmixture with the finely divided active compound. In the tablet theactive compound is mixed with carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired. The powders and tablets preferably contain from 0.1 to 1.0 toabout 70 percent of the active ingredient. Suitable solid carriers aremagnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.The term "preparation" is intended to include the formulation of theactive compound with encapsulating material as carrier providing acapsule in which the active component with encapsulating material ascarrier providing a capsule in which the active component (with orwithout other carriers) is surrounded by carrier, which is thus inassociation with it. Similarly, cachets are included. Tablets, powders,cachets, and capsules can be used as solid dosage forms suitable fororal administration.

For preparing suppositories, a low melting wax such as a mixture offatty acid glycerides or cocoa butter is first melted, and the activeingredient is dispersed homogeneously therein as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions.As an example may be mentioned water or water-propylene glycol solutionsfor parenteral injection. Liquid preparations can also be formulated insolution in aqueous polyethylene glycol solution. Aqueous solutionssuitable for oral use can be prepared by dissolving the active componentin water and adding suitable colorants, flavors, stabilizing, andthickening agents as desired. Aqueous suspensions suitable for oral usecan be made by dispersing the finely divided active component in waterwith viscous material, i.e., natural or synthetic gums, resins,methylcellulose, sodium carboxymethylcellulose, and other well-knownsuspending agents.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions, and emulsions. These particular solid form preparations aremost conveniently provided in unit dose form and as such are used toprovide a single liquid dosage unit. Alternately, sufficient solid maybe provided so that after conversion to liquid form, multiple individualliquid doses may be obtained by measuring predetermined volumes of theliquid form preparation as with a syringe, teaspoon, or other volumetriccontainer. When multiple liquid doses are so prepared, it is preferredto maintain the unused portion of said liquid doses at low temperature(i.e., under refrigeration) in order to retard possible decomposition.The solid form preparations intended to be converted to liquid form maycontain, in addition to the active material, flavorants, colorants,stabilizers, buffers, artificial and natural sweeteners, dispersants,thickeners, solubilizing agents, and the like. The liquid utilized forpreparing the liquid form preparation may be water, isotonic water,ethanol, glycerine, propylene glycol, and the like as well as mixturesthereof. Naturally, the liquid utilized will be chosen with regard tothe route of administration, for example, liquid preparations containinglarge amounts of ethanol are not suitable for parenteral use.

Preferably, the pharmaceutical preparation is in unit dosage form. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself or it can be the appropriate number of any of these inpackaged form.

The quantity of active compound in a unit dose of preparation may bevaried or adjusted from 0.01 mg to 50 mg preferably to from 0.5 to 10 mgaccording to the particular application and the potency of the activeingredient. The compositions can, if desired, also contain othercompatible therapeutic agents.

In therapeutic use, the mammalian dosage range for a 70 kg subject isfrom 0.1 to 150 mg/kg of body weight per day or preferably 0.2 to 75mg/kg of body weight per day. The dosages, however, may be varieddepending upon the requirements of the patient, the severity of thecondition being treated, and the compound being employed. Determinationof the proper dosage for a particular situation is within the skill ofthe art. Generally, treatment is initiated with smaller dosages whichare less than the optimum dose of the compound. Thereafter the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. For convenience, the total daily dosage may bedivided and administered in portions during the day if desired.

We claim:
 1. The compound designated CL-1724B-2 possessing antimicrobialactivity; compound CL-1724B-2 characterized by:a molecular weight of1374 atomic mass units; an elemental analysis corresponding to 52.4% C,6.26% H, 4.08% N, 9.32% S, 27.94% O; an ultraviolet absorption spectrumin methanol showing absorption maxima at 318 nm (a=8.12), 280 nm(a=11.16), 251 nm (a=18.64), and 204 nm (a=29.84); an infraredabsorption peaks at 3520, 2940, 1760, 1720, 1685, 1600, 1580, 1520,1450, 1385, 1375, 1310, 1250, 1155, 1075, 1015, 1000, 955, 900, 875, and850 reciprocal centimeters; a 300 MHz proton magnetic resonance spectrumin deuterochloroform exhibiting principal signals at 0.90 (multiplet),1.25 (singlet), 1.40 (multiplet), 1.45-2.20 (multiplet), 2.30(multiplet), 2.40-2.90 (multiplet), 3.30-4.30 (multiplet), 4.50(multiplet), 4.70 (doublet of doublets), 4.95 (doublet), 5.35 (singlet),5.45 (doublet), 5.70 (multiplet), 5.80 (doublet), 5.90 (doublet), 6.25(singlet), 6.55 (multiplet), 7.75 (singlet), 8.65 (singlet), 12.95(singlet), all signals measured in parts per million downfield fromtetramethylsilane.
 2. A pharmaceutical composition comprising anantimicrobially effective amount of CL-1724B-2 compound as defined inclaim 1 in combination with a pharmaceutically acceptable carrier.
 3. Aprocess for the production of CL-1724B-2 compound as defined in claim 1which comprises cultivating a strain of actinomycete NRRL 15758 underaerobic conditions in a culture medium containing assimilable sources ofcarbon and nitrogen until a substantial amount of CL-1724B-2 compound isproduced and subsequently isolating said compound.
 4. A method oftreating microbial infections in a mammal comprising administering to amammal in need of such treatment an antimicrobially effective amount ofcompound CL-1724B-2 as defined in claim 1 in combination with apharmaceutically acceptable carrier.